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. 2020 Aug 30;17(11):1603–1612. doi: 10.1080/15476286.2020.1813439

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This work was authored as part of the Contributor‘s official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 U.S.C. 105, no copyright protection is available for such works under U.S. Law.

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Figure 3. — Drosha-AS27a and Drosha-AS32a are deficient in miRNA biogenesis.

(A) Schematic diagram of experimental workflow for investigation of Drosha-AS27a and Drosha-AS32a function. (B) Northern blot to analyse the function of Drosha-AS27a and Drosha-AS32a in miR-16 processing. CMV (Pol II) driven pri-miR-16 was ectopically expressed in HEK293T cells, as well as co-expressed with various Drosha variants in the Drosha KO cells. Pri-miRNA-16 transcripts are too long to run into the polyacrylamide gel. Pre-miR-16 and mature miR-16 were detected by a probe against the sequence of miR-16. The expression of U6 served as a loading control. (C) Levels of endogenous miRNAs were measured by small RNA sequencing in HEK293T cells and Drosha KO cells rescued with Drosha-FL, Drosha-AS27a, or Drosha-AS32a. For each miRNA, the average level of two biological replicates was calculated. miRNAs with an average expression >10 reads per million (RPM) in WT HEK293T cells were plotted.